Adenovirus

OVERVIEW: What every clinician needs to know

Pathogen name and classification

Adenovirus is a double-stranded DNA virus in the family Adenoviridae.

Human adenoviruses are divided into six species (A-F). Individual serotypes are designated numerically (e.g., Ad5, Ad7). Clinical syndromes are associated with specific serotypes, although there is wide geographic and temporal variation in strains resulting in outbreaks of adenovirus infection.

What is the best treatment?

There are no FDA-approved agents for the treatment of adenovirus infections. The most effective, clinically available antiviral agent active against adenovirus is cidofovir. Cidofovir is also the drug with which there is the most published experience in severe infections in immunocompromised patients. The role of antiviral therapy for adenoviral infections depends greatly on the clinical setting. There are several broad categories of adenoviral infection, which can be defined as mainly involving specific organ systems (i.e., respiratory disease, ocular disease, gastrointestinal disease, genitourinary disease, central nervous system disease, and disease in immunocompromised patients.

Cidofovir, an acyclic nucleoside analog, inhibits adenoviral DNA polymerase and causes termination of DNA synthesis. The major toxicity of cidofovir is nephrotoxicity. There are no randomized, controlled studies of the efficacy of cidofovir in the treatment of adenoviral infection. However, there are numerous case reports and series of immunosuppressed patients treated successfully with cidofovir. Specific organ systems commonly involved by adenovirus infection in the immunosuppressed patient include the respiratory, hepatic, and urinary systems. Colitis, ocular involvement may also occur. Treatment with cidofovir at 5mg/kg given once weekly is the recommended dosage for invasive and disseminated disease, although dose-limiting toxicity in the patient receiving multiple nephrotoxic drugs is common. The nephrotoxicity of cidofovir treatment may be ameliorated by adequate hydration and co-administration of probenecid, although limited data exist on the protective effect of probenecid. Some of these studies have also used ribavirin either in conjunction or as a prophylactic agent prior to the development of overt adenovirus infection.

The primary risk factors for development of persistent adenovirus viremia and invasive disease are low T lymphocyte counts and treatments or protocols that directly lower T lymphocyte counts, including anti-thymocyte globulin, T-cell-depleted grafts, alemtuzumab, and prolonged immunosuppression. Persistent and increasing adenovirus levels in peripheral blood are correlated with an increased risk of invasive disease and mortality. In patients at high risk of adenovirus disease, pre-emptive strategies have been advocated. Adenovirus levels are monitored weekly in peripheral blood by quantitative polymerase chain reaction (PCR), and pre-emptive therapy with cidofovir instituted when the viral load exceeds pre-defined cutoff levels. The level at which empiric pre-emptive therapy should be employed is not clear-cut, as the risk of invasive disease is highly influenced by accompanying risk factors. In the highest risk patients, adenovirus levels as low as 100 copies/ml have been used as a threshold value for pre-emptive treatment, whereas 10,000 copies/ml have been used as a cut-off value in lower risk patients. It should be noted that the presence of circulating adenoviral DNA may be present in healthy individuals, albeit less commonly and at lower levels.

The decisions regarding pre-emptive treatment with cidofovir in this situation is similar to the decision regarding CMV treatment in transplant recipients in whom a rapidly rising viral load is correlated with an increased risk of disease but the decision to institute treatment is based on host risk factors, as well as the absolute viral load. In addition, extrapolation of results based on viral load across institutions is difficult because of variations in testing methodology and sensitivity among laboratories. Validation of PCR test performance characteristics and correlation with institution-specific outcomes is, therefore, critical in therapeutic decisions based on adenovirus PCR measurements.

Lower dose cidofovir (1 mg/kg) three times a week has been advocated to limit nephrotoxicity, especially when administered as pre-emptive therapy based on viral load alone in the absence of disease symptoms. We have successfully used this lower dose regimen with acceptable toxicity and suppression of viremia in the setting of HSCT (unpublished observations). The length of therapy, either in the pre-emptive setting, or as treatment of active disease remains unestablished. It is clear, however, that the patient remains at significant risk until T-cell counts recover, especially adenovirus-specific cell-mediated immunity. Thus, reduction of iatrogenic immunosuppression, to the extent that it is possible, is a critical aspect of successful treatment of adenoviral infection in the immunosuppressed patient. It is important to note that, although cidofovir has activity against many herpes viruses and is used as a salvage therapy for CMV infection, the lower dose regimen of 1 mg/kg three times a week for adenovirus may be insufficient to treat or prevent CMV reactivation or disease in the immunosuppressed patient. Thus, CMV disease has supervened in patients undergoing treatment for adenovirus with the thrice weekly cidofovir regimen.

Although topical cidofovir has been effective in animal models of adenoviral ocular disease, toxicity in human trials has led to abandonment of its development for treatment of conjunctivitis. As mentioned, although ribavirin has been used as treatment of adenoviral infections, its clinical success has been variable. In addition, ribavirin is inactive against several serotypes of adenovirus, and its use is, therefore, not generally recommended for adenoviral infections.

Development of resistance to cidofovir has been documented in animal models, but its relevance to clinical failure of treatment has not been demonstrated. As previously described, the primary determinant of successful treatment is recovery of immune function, and the role of cidofovir is to suppress adenoviral replication until immune reconstitution occurs.

Testing of adenovirus isolates for drug resistance is not routinely performed.

Alternative therapies have focused on the delivery of T-cells with activity against adenovirus. Such therapies can be broadly categorized as employing unselected donor lymphocytes, populations of donor lymphocytes selectively enriched for activity against adenovirus, or cytotoxic T lymphocytes generated by stimulation with adenovirus epitopes ex vivo. Although donor lymphocyte infusions have been reportedly successful in refractory cases of adenoviral infection, the attendant complications of graft versus host disease (GVHD) have limited its utility. Adenovirus-specific T lymphocytes derived after stimulation in vitro with adenovirus and selected based on cytokine secretion have been demonstrated to reduce adenovirus loads in blood. An alternative approach that has successfully been used against EBV-associated lymphoproliferative disease has recently been expanded to include treatment of adenovirus infections in HSCT patients. Cytotoxic T lymphocytes (CTL) lines are generated and expanded in vitro by incubation with adenovirus transduced cells or cells in which adenovirus proteins have been expressed by transfection. Infusion of these CTL lines has led to reduction of adenoviral loads in several patients and successful treatment of refractory disease. The limitations of cost, labor, and time required to generate individual CTL lines for individual patients limit the availability of such therapies to highly specialized research treatment facilities. However, technological advances are decreasing, such barriers to the more general availability of such treatment modalities.

A lipid-conjugated form of cidofovir (hexadecyloxypropyl cidofovir) is currently undergoing clinical trials for a variety of viral infections, including adenovirus. The ability of the lipid conjugate to undergo rapid cellular translocation in the gut and into target cells prior to cleavage to the active form of the drug permits oral administration and may limit nephrotoxicity.

How do patients contract this infection, and how do I prevent spread to other patients?

Epidemiology

The epidemiology of adenovirus infection is highly dependent on the age of the populations studied. Specific serotypes affect primarily children, with adults remaining more susceptible to other, distinct serotypes. Adenovirus is shed in the stool for prolonged periods, particularly in children who may shed virus asymptomatically for months. Adenoviral infection may be asymptomatic, complicating the attribution of specific clinical syndromes to viral isolates. However, specific serotypes (e.g., 40 and 41) are associated with outbreaks of diarrheal illness, which are transmitted by the fecal-oral route. Epidemic outbreaks of adenovirus associated respiratory disease, primarily pneumonia, have long been recognized in military recruits. Such episodes classically occur with the onset of cold weather and primarily in those without prior exposure, hence the high incidence in new recruits. Further, the restriction of such epidemics to barrack-type conditions suggests that close contact and possibly stress and fatigue contribute to efficient transmission. Respiratory disease occurs primarily by droplet transmission of infectious secretions.

Conjunctivitis may be spread from person-to-person or via fomites and has also been associated with contact with contaminated water, such as in swimming pools. Epidemic keratoconjunctivitis is a distinct entity, which is highly contagious and is linked to specific serotypes, such as Ad37.

Adenovirus infections occur throughout the world and are ubiquitous in all populations, although transmission is enhanced by conditions of poor hygiene and overcrowding. Hemorrhagic cystitis occurs more commonly in males.

The incidence of adenovirus respiratory infection in military populations increased during periods when recruits were no longer vaccinated. The overall incidence of adenovirus infection in the general population has not undergone any obvious changes; however, clustered outbreaks among children occur worldwide.

Infection control issues

Droplet precautions and strict hand hygiene should be employed to prevent nosocomial transmission of adenovirus.

A vaccine against serotypes 7 and 4 was generally available for the military until production ceased in 1999. The vaccine has recently been re-approved for use in military populations between the ages of 17 and 50. The vaccine is an oral formulation containing live adenovirus. Two tablets, one containing Ad4 and the other containing Ad7, are administered as a single dose. The coating results in infection of the intestinal tract, bypassing the respiratory system, and results in seroconversion. The vaccine is not attenuated; therefore, adenovirus vaccine strains are shed in the stool for up to 28 days following administration. The vaccine is not approved for use in pregnant women. However, four pregnancies occurred during field testing of the vaccine and none of the women or children displayed any adverse effects from the vaccine. An adenovirus vaccine is not currently available for civilian use.

The major protective mechanisms against adenovirus infection are cell mediated. Both CD4+ and CD8+ T lymphocytes specific for adenovirus epitopes are demonstrable in humans. CD4+ T lymphopenia is a major risk factor for invasive infection in the immunocompromised host, and return of CD4+ T cells correlates with recovery from infection. Innate immune responses are clearly elicited by adenovirus infection, and interferon secretion is induced by adenovirus infection. Several adenovirus genes modulate the host immune response by a variety of mechanisms. E1A protein inhibits IFN signal transduction. Adenovirus VA RNAs, small noncoding RNAs block host translational shutoff that normally occurs as a response to viral infection. E3 proteins interfere with host antigen presentation, chemokine release, and apoptosis of infected cells. Consistent with the function of these adenovirus genes, the most robust inflammatory responses are often seen in patients or in animals infected with adenovirus vectors in which adenovirus genes with immunodulatory function have been deleted.

Clinically, the major risk factors for infection are various types of immunosuppression. Although all patients with congenital and acquired forms of immunodeficiency are at increased risk, specific conditions appear to be more potent risk factors. Among HSCT patients, matched unrelated donor transplants and immunosuppressive therapy, such as anti-thymocyte globulin (ATG) and alemtuzumab, are associated with increased risk. Graft versus host disease has been associated with disseminated adenovirus infection, but whether GVHD actually predisposes to adenovirus disease is unclear. Risk factors for adenovirus disease in solid organ transplantation (SOT) include pediatric transplantation, anti-lymphocyte globulin therapy, liver and small bowel transplantation, and donor-positive, recipient-negative adenovirus serology.

Early in infection, eosinophilic nuclear inclusions are present with a characteristic clear halo. Smudge cells are a characteristic finding of cells in the late stage of infection. In smudge cells, the nuclear membrane is indistinct and the nucleus is almost completely occupied by a large round or oval basophilic inclusion.

What are the clinical manifestations of infection with this organism?

Respiratory disease in children: Adenoviral respiratory disease consists of a broad spectrum of clinical manifestations, ranging from a mild upper respiratory illness to frank pneumonia. Respiratory symptoms may be accompanied by pharyngitis and lymphadenopathy. Although generally mild, outbreaks of pneumonia with high mortality in children have been reported. In some series of hospitalized children with documented adenoviral infection high fever, leukocytosis and exudative pharyngitis resembling bacterial infection has been observed. Ad3 and recombinant strains have been associated with severe outbreaks of adenoviral respiratory infection with fatalities in young children. Pharyngoconjunctival fever is a syndrome characterized by conjunctivitis, pharyngitis, fever, and preauricular and cervical lymphadenopathy. Respiratory symptoms may be absent, and the infection may present as isolated conjunctivitis. Acute respiratory disease (ARD) in clustered settings: ARD is also comprised of a spectrum of disease, ranging from relatively mild upper respiratory disease to frank pneumonia. Symptoms during outbreaks among military recruits have ranged from pharyngitis, cough, and fever to bronchitis and pneumonitis. Ad4, Ad7, and Ad14 have been most commonly associated with outbreaks among military recruits. Disease severity in this setting can be significant, leading to hospitalization of more than 40% of symptomatic patients.

Ocular disease

Acute conjunctivitis: Adenoviral conjunctivitis presents as unilateral or, more commonly, bilateral follicular conjunctivitis. Transmission may occur from contacts and fomites or from common source exposure to water sources, such as swimming pools or fresh water ponds. The disease is generally benign and self-limited, resolving in less than 7 days without sequelae. Conjunctivitis has most commonly been associated with Ad3 and Ad7.

Epidemic keratoconjunctivitis (EKC): The syndrome of EKC is clinically distinct from the benign adenoviral conjunctivitis common in children. EKC is characterized by more severe symptoms, including pain, lacrimation, and photophobia. Involvement is frequently unilateral but may spread to involve both eyes. Inflammation may be extensive, resulting in subcorneal opacities and scarring with visual impairment. EKC is highly contagious and has been associated with Ad8, Ad19, Ad37, and, less commonly, other serotypes.

Genitourinary disease: Hemorrhagic cystitis caused by adenovirus is characterized by gross hematuria in the absence of other identifiable causes and adenoviral detection in urine and blood. Hemorrhagic cystitis occurs primarily in young males, for unknown reasons. Hemorrhagic cystitis is associated with serotypes 11 and, to a lesser degree, 21. Gross hematuria occurs for 2-5 days, accompanied by dysuria, but is self-limited and resolves without sequelae. The severity and complications of hemorrhagic cystitis due to adenoviral infection in immunocompromised patients are, however, much greater.

Gastrointestinal disease: Ad40 and Ad41 are the predominant strains that have been implicated as significant causes of infantile diarrhea. The major symptom is diarrhea, which may sometimes be prolonged. Prospective studies have shown that a significant number of infections occurring during outbreaks may be asymptomatic.

Infections in immunocompromised patients: Adenovirus infection is a particularly difficult problem in HSCT and SOT patients, particularly those with additional risk factors resulting in impaired T-cell function. Adenovirus disease is also more likely in the immunosuppressed pediatric population and correlates with a higher risk of primary infection and reactivation. Disseminated infection may involve virtually any organ system, resulting in clinically significant hepatitis, pneumonia, hemorrhagic cystitis, or colitis. Infection in the transplanted organ is particularly likely in SOT recipients.

Respiratory disease: Adenovirus pneumonia is typically presents like other viral pneumonias with interstitial infiltrates. The cough is generally nonproductive in the absence of bacterial superinfection. Adenoviral pneumonia in the lung transplant recipient has been histopathologically demonstrated to result in bronchocentric necrosis, hemorrhage, and cellular infiltration. Multiple cell types (pneumocytes, macrophages, and bronchial epithelial cells) demonstrated evidence of adenovirus infection with characteristic nuclear inclusions (smudge cells). Adenovirus colitis may present in immunosuppressed adults as an inflammatory gastroenteritis with epithelial sloughing and goblet cell infection, with typical inclusions in scattered cells.

Hepatic disease: Adenovirus hepatitis has the worst prognosis in liver transplant recipients but may also occur in HSCT patients. Although more common in pediatric patients, adenoviral hepatitis also occurs in adult liver transplant recipients and has a poor prognosis. A pattern of coagulative necrosis has been described in adenoviral hepatitis. A lack of significant inflammation may be more characteristic of adenoviral etiology than GVHD or rejection.

How should I identify the organism?

Tissue samples from the affected organ are generally of highest utility. Respiratory secretions, nasopharyngeal swabs are useful in diagnosing respiratory and pharyngeal infection. Conjunctival scrapings or swabs may be examined in conjunctivitis. Detection of virus in stool may be useful for epidemiological investigation but is often positive for months after acute infection because of asymptomatic shedding. In immunocompromised patients, biopsy specimens may be very useful in documenting the cause of hepatitis or colitis, allowing identification of the virus or diagnostic histopathologic changes in infected cells. Bronchoscopic specimens may be examined in cases of pneumonitis in immunocompromised patients. Serial quantitative determination of adenovirus viral load in peripheral blood by PCR is extremely useful in monitoring high-risk SOT and HSCT patients.

Adenovirus may be detected directly from respiratory, ocular, or urinary samples by fixing cells, staining with antibodies, and examining the cells by fluorescence microscopy. Although rapid, the direct antigen testing is generally less sensitive (~60%) than culture. Electron microscopy reveals arrays of virions in adenovirus-infected cells but is not used outside of the research setting.

Adenovirus may be cultured by inoculation onto laboratory-grown human or xenogeneic cell lines. Growth is usually optimal on human-derived cells. Growth of some serotypes is best on HEK 293 cells, which are transformed by adenovirus genes that may provide helper function for the infecting virus. Growth is confirmed by staining for adenovirus antigens.

Adenovirus can be cultured on a variety of human cell lines. The shell vial technique involves staining of infected cell monolayers with virus-specific antibody 48 hours after infection.
Cytopathic effect (CPE) can be evident within 2 hours but may take weeks, depending on inoculum size and other factors. Infected cells swell, round up, detach from the plate, and ultimately undergo lysis.
Serotyping is not routinely performed but may be done for research studies. PCR and sequencing may also be used to further characterize and classify adenovirus genotypes.
The replicative cycle occurs over 24-36 hours in vitro, but clinical detection by conventional culture may take days or weeks.
Overall culture techniques are highly sensitive and are traditionally the gold standard for adenovirus detection. However, cultures must be held for extended periods to achieve full sensitivity.

Quantitative PCR is now commercially available to measure adenovirus loads in blood. Such testing may also be performed on other body fluids, but standardization of such testing is problematic and the interpretation of results may be complex. Both sensitivity and specificity are high when performed in experienced laboratories. PCR techniques are specific for the genomic sequences represented in the primers used and, therefore, may be limited in the range of strains they are capable of detecting.

Enzyme-linked immunosorbent assay (ELISA) testing is available and is rapid but less sensitive than culture.

How does this organism cause disease?

Adenovirus encoded proteins that may be important in immune evasion, protection of infected cells from apoptosis, and preventing host cell shutoff have been described

The determinants of tissue-preferential pathogenesis displayed by different strains (e.g. the propensity of Ad 40/41 to cause GI disease) are uncharacterized and unlikely to be based on specific cell tropism, as the receptors for adenovirus are not known to be highly cell-type specific.

WHAT’S THE EVIDENCE for specific management and treatment recommendations?

Berk, A, Knipe, DM, Howley, PM. “Adenoviridae: the viruses and their replication”. Fields virology. vol. 2. 2007. pp. 2355-94. (A comprehensive review of the virology, epidemiology, and pathology of adenovirus infections in immunocompetent and immuno-compromised patients.)

Leen, AM, Christin, A, Myers, GD. “Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation”. Blood. vol. 114. 2009. pp. 4283-92. (A description of new alternative cytotoxic T cell therapy directed specifically against adenovirus and other viral infections in transplant recipients.)

Lindemans, CA, Leen, AM, Boelens, JJ. “How I treat adenovirus in hematopoietic stem cell transplant recipients”. Blood. vol. 116. 2010. pp. 5476-85. (A comprehensive discussion of strategies to stratify risk for adenovirus infection and therapeutic algorithms for high-risk HSCT patients.)

Wold, W, Horwitz, M, Knipe, DM, Howley, PM. “Adenoviruses”. Fields virology. vol. 2. 2007. pp. 2395-436. (A comprehensive review of the virology, epidemiology, and pathology of adenovirus infections in immunocompetent and immunocompromised patients.)

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